Unraveling defects in the human innate immune response: Diagnosis of inherited disorders in the NF-κB pathway

Primary immunodeficiencies are inherited diseases that predispose children and adults to recurrent bacterial, viral, and/or fungal infections. The awareness for primary immunodeficiencies is still limited in routine clinical practice and many patients are diagnosed too late to prevent life-threatening infections by appropriate therapy. Toll-like/IL-1R (TIR) receptors and the associated NF-κB signaling pathway are important mediators of the innate immune response. Understanding their function in human cells is necessary as defects lead to invasive pyogenic bacterial infections, herpes simplex encephalitis, common variable immunodeficiency, chronic mucocutaneous candidiasis, and combined immunodeficiency.

In the first part of the study, we determined the value of TLR/NF-κB-specific functional assays to diagnose these disorders.

In Chapter 1, we studied induction of IL-6 by PBMCs after 24 hour stimulation with various TIR ligands in 770 patients presenting with increased susceptibility to infection. We established cut-off values for IL-6 induction by PBMCs after stimulation with various TIR ligands by applying the Bhattacharya algorithm. Application of the established cut-off values resulted in the identification of 22 patients with impaired induction of IL-6 after TIR stimulation, including one IRAK-4 deficient and two NEMO-ID patients. The other 19 patients presented with a phenotype reminiscent of a mild IRAK-4 or MyD88 deficiency, but without mutations in these genes.

Chapter 2 focused on the development of a rapid flow cytometric assay that can screen for genetic defects in the canonical NF-κB pathway. In terms of validation, this assay based on the evaluation of IκB-α degradation can already be used to screen for IRAK-4, MyD88-, and CARD11-deficiencies. It remains to be determined how patients with other defects in the canonical NF-κB pathway respond (study still ongoing in collaboration with Hôpital Necker, Paris).

Despite the usefulness of functional assays, genetic analysis is still the only way to accurately establish a diagnosis of NF-κB immunodeficiencies. In the second part, we explored the currently available genetic methods that can be used to identify disease-causing mutations in the innate immune system.

In Chapter 3, we set up a highly reliable parallel resequencing technique to sequence IKBKG, IRAK4, and MYD88 for several patients in one run by comparing Single Molecule Real Time (SMRT) sequencing on the PacBio RSII (Pacific Biosciences) with Sequencing-by-Synthesis on the Hiseq 2500 (Illumina). We targeted IKBKG specifically as it is one of the genes with insufficient coverage due to the presence of the highly homologous pseudogene IKBKGP1.

Chapter 4 describes three patients who obtained a genetic diagnosis through Sanger sequencing. We describe two patients in whom an IKBKG mutation was found. In a third patient, carrying no IKBKG variants, a gain-of-function STAT1 mutation was identified. We studied pathologic impact of the identified mutations and their contribution to the immunological phenotype in all three cases.

Chapter 5 describes the next-generation sequencing application of an array-based panel covering 174 PID genes that has been implemented in our center since 2014. This technique was used to diagnose IRAK-4 deficiency in an adult woman who was originally thought to suffer from autosomal dominant hyper-IgE syndrome.

In Chapter 6, patients in whom an innate immune defect was suspected, were subjected to whole exome sequencing (WES) to discover yet unappreciated disease-causing gene candidates. A total of 25 patients with recurrent pyogenic bacterial infections were evaluated with WES. In five of them, mutations were found in the CVID-associated TNFRSF13B gene. Two patients with an unknown syndromic immunodeficiency were also subjected to whole exome sequencing. The first patient suffered from X-linked Kabuki syndrome caused by a novel hemizygous de novo mutation in KDM6A. A homozygous mutation in TRNT1 was identified in the second patient leading to a severe immunodeficiency characterized by gastro-intestinal inflammation and B-cell deficiency. 

In conclusion, we evaluated diagnostic strategies that can be used in the clinical and genetics laboratory for a specific subset of primary immunodeficiencies affecting the innate immune system.